It is proposed that nitric oxide (NO) is a critical messenger and effector molecule in skin physiology and homeostasis and that altered levels of NO in the skin cause disease. The goal of the proposal is to define the role of NO in skin with an emphasis towards determining the functional effects of this gas. Human, but not murine, keratinocytes have been demonstrated to have the capacity to express inducible form of nitric oxide synthase (iNOS) and produce NO. This critical difference between mice and men may explain why mice do not develop cutaneous eruptions analogous to those found in humans. Transgenic mice in which NOS is targeted to the skin mimic are shown here to develop phenotype found in human conditions. In conventional mice, iNOS is expressed in Langerhans cells (LC). NO is also produced by the LC-like cell line XS-52. NO produced in LC may affect LC function and, as NO is freely diffusible across cell membranes, it has the capacity to affect adjacent cells in the epidermis. Toxic effects of NO on melanocytes and keratinocytes suggest that NO may be an effector molecules in a number of skin conditions, including post-inflammatory hypo-pigmentation, vitiligo, graft versus host disease (GVH), scleroderma and the often fatal process, toxic epidermal necrolysis (TEN). In the proposed studies, purified LC will be evaluated for the production of iNOS using RT-PCR and measurement of NO using the Griess reaction and the conversion of radioactive L-arginine to citrulline. The effects of selected cytokines in the regulation of NOS will be examined. The effects of NO on XS-52 cells themselves will be studied. The mechanism of NO- induced killing of melanocytes and keratinocytes will be examined in co- culture experiments with XS-52 cells and via incubation with NO donors. Transgenic mice in which NOS expression is targeted to the epidermis have been produced, phenotypically develop white hair and histopathologically, scleroderma. These animals will be characterized further. Mice expressing NOS under the control of an inducible promoter will be generated. The transgenic mice will be tested as models for disorders of pigmentation, antigen presentation, scleroderma, TEN and GVH. These results have the potential to lead to novel therapeutic strategies for the treatment of human disease.